Investigations of the biosynthesis of two novel microbial metabolites will be continued. The first is the antitumor agent valanimycin, an azoxy compound produced by S. viridifaciens. This compound is important because its damages DNA, and because it containan N-N bond. Although natural products containing N-N bonds are widely distributed, little is known about the mechanism(s) of N-N bond formation. Previous studies of valanimycin biosynthesis revealed some steps in the biosynthetic pathway and led to the isolation of a cosmid containing all of the genes required for valanimycin biosynthesis. Future studies of valanimycin have several goals. The first is to finish the sequencing and analysis of the genes required for valanimycin production. The second is to identify the gene(s) required for N-N bond formation by sequence analysis, and non-polar gene disruptions. The third is to overproduce the protein(s) required for N-N bond formation and examine the N-N bond forming chemistry in vitro. The fourth is to further characterize putative valanimycin resistance gene. The fifth goal is to complete studies of the two-component flavin monooxygenase from the valanimycin pathway that catalyzes the oxidation of isobutylamine to isobutylhydroxylamine. The last goal is to identify function of the other genes in the valanimycin cluster. The second metabolite to be investigated is the antitumor agent sparsomycin, a potent inhibitor of protein biosynthesis produced by S. sparsogenes. Sparsomycin is important because it inhibits the peptidyltranserase step in protein biosynthesis by interaction with 23S rRNA and because its biosynthesis involves novel biochemical reactions including the oxidative insertion of sulfur into a C-H bond, and the oxidative cleavage of both rings of the indole nucleus of tryptophan. Previous studies of sparsomycin revealed the outlines of the biosynthetic pathway and led to the isolation of a cosmid that confers sparsomycin resistance and carries the gene encoding an enzyme ("UCA synthase") involved in sparsomycin biosynthesis. Future studies of sparsomycin biosynthesis have several goals. The first is to sequence and characterize the gene(s) for sparsomycin self-resistance that reside on the isolated cosmic second is to localize and sequence the gene encoding UCA synthase. The third goal is to overproduce UCA synthase and carry out additional mechanistic studies with the enzyme. The fourth is to locate the genes encoding the proteins responsible for the oxidative insertion of sulfur and the oxidative opening of the indole nucleus. The final goal is to overproduce and study the sulfur insertion and dioxygenase proteins in vitro.